The present invention relates to a polypeptide construct comprising at least one CDR3 region, wherein at least one of said at least CDR3 regions comprises at least one substitution in the amino acid sequence YYDDHY (SEQ ID NO.1) and wherein said at least one substitution comprises: in the first position of SEQ ID NO:1 a substitution from Y to H; in the second position of SEQ ID NO. 1 a substitution from Y to S, from Y to N, from Y to F or from Y to H; in third position of SEQ ID NO. 1 a substitution from D to N or from D to E; in the forth position of SEQ ID NO. 1 a substitution from D to Q, from D to A, from D to V, from D to E or from D to G; in the fifth position of SEQ ID NO. 1 a substitution from H to Q, from H to P, from H to Y, from H to R or from H to N; or in the sixth position a substitution from Y to N.
Furthermore, the invention provides for polynucleotides encoding said polypeptides as well as for vectors and host cells comprising said polynucleotides. Additionally, the invention relates to compositions, preferably pharmaceutical or diagnostic compositions comprising the polypeptides, polynucleotides, vectors or host cells of the invention.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated by reference.
T cells are a major population of lymphocytes. These cells express a unique membrane bound antigen-binding molecule, the T-cell receptor (TCR). The T-cell receptor associates with the cluster of differentiation “CD3”, forming the TCR-CD3 membrane complex.
Despite advances in understanding how T cells are activated (Davis, 1998, Annu. Rev. Immunol. 16, 523-44) and how the T cell signal is propagated within the cell (Germain, 1999, Annu. Rev. Immunol. 17, 467-522), few details are known of the mechanism by which engagement of a TCR by its ligand results in signaling.
A number of models have been proposed concerning the mechanism of T-cell activation and signal transduction (Davis, 1998, Annu. Rev. Immunol. 16, 523-44; Baker 2001, Immunity 14, 681-692). These include oligomerization of TCR-peptide/MHC complexes (Bachmann, 1999, Immunol. Today 20, 568-576), serial triggering (Vallitutti, 1995, Nature 375, 148-151; Viola, 1999, Cell 96, 1-4), conformational changes occurring within a single TCR heterodimer (Janeway, 1995, Immunol. Today 16, 223-225), geometrical rearrangements within a multivalent TCR/CD3 complex (Ding, 1999, Immunity 11, 45-56), and segregation of kinase and phosphatases due to varying sizes of extracellular domains (van der Merwe, 2000, Semin. Immunol. 12, 5-21). In each model, the molecular event (e.g. oligomerization or conformational change) is presumed to alter the degree of phosphorylation on the cytoplasmic side of the membrane in favor of signal transduction.
When defined oligomers of MHC class II-peptide complexes were used to trigger T cells, it was found that at least three TCRs need to be brought together in order to induce a calcium response (Boniface, 1998, Immunity 9, 459-466). In another study T cell activation could be induced either by dimers of MHC class I-peptide complexes or by monomers that cross-link the TCR with the CD8 coreceptor-Lck kinase complex (Delon, 1998, Science 281, 572-575).
The serial triggering model proposes that a small number of peptide/MHC complexes can cause activation by transiently binding many TCRs (Viola, 1996, loc. cit.). The actual measurements have been difficult, however, partly because the membrane-bound nature of MHC molecules and TCRs required the engineering of soluble forms, partly because the affinities are only measurable with highly sensitive technology.
The first measurements of TCR affinities for peptide/MHC complexes were made by Matsui 1991, Science 254, 1788-1791 and Weber 1992, Nature 356, 793-796, which showed low affinity binding KD˜10-50 μM. Later experiments (Sykulev 1994, Immunity 1, 15-22; Sykulev 1996, Immunity 4, 565-571) used a different experimental approach and showed higher affinities KD˜0.1 μM.
Recently the development of surface plasmon resonance instruments, particularly the BIAcore™ (Pharmacia Biosensor) allowed measuring the kinetics of TCR binding to the peptide/MHC complex. In some cases the fast off-rate measured was significantly stabilized if soluble CD8 was introduced (Garcia 1996, Nature 384, 577-81). This indicates that T cell activation might involve the interaction of additional components like CD8 with the peptide/MHC complex, although recognition of the peptide/MHC complex is mediated solely by the TCR-CD3 complex.
The TCR-peptide/MHC complex interaction as well as the interaction of additional T cell components with the peptide/MHC complex may require an optimal dwell time (Kalergis 2001, Nature Immunol. 2, 229-234; Viola 1996, loc. cit.) and may have an effect upon the kinetics of T cell activation. Half lives for the interaction complexes were measured between 10-30 sec (Davis 1998, loc. cit.).
In contrast, data on dissociation rates measured by BIAcore™ do not support the serial triggering model (Davis 1998, loc. cit.). This is because, thus far, all improvements in TCR/peptide/MHC complex stability within any one system result in a more robust T cell response (Davis et al. 1998, Annu. Rev. Immunol. 16, 523-44) rather than exhibiting a normal distribution around some optimum value as proposed (Kalergis 2001, Nature Immunol. 2, 229-234). In summary, it has to be emphasized that to date, the data from TCR-peptide/MHC interactions available in the literature do not preferentially support any one T cell activation model.
It has been known since the early 1980s that T cell activation can also be induced by anti-TCR antibodies. Recent data on antibody-induced T cell activation have provided support for oligomerization of TCRs upon ligand contact (Reich 1997, Nature 387, 617-20; Brown 1993, Nature 364, 33-39), although there are dissenting views.
Some experiments have used bispecific reagents for T cell targeting (Traunecker 1991, Embo J. 10, 3655-3659; Mack 1995, PNAS 92, 7021-7025; Mack 1997, J. Immunol. 158, 3965-3970). Bispecific antibodies can be used for the binding to the TCR/CD3 complex and to a cell surface antigen to target cytotoxic T lymphocytes against a target of choice (Staerz 1985, Nature 314, 628-631; Lanzavecchia 1987, Eur. J. Immunol. 17, 105-111). The monovalent binding to CD3 does not result in T cell activation. Therefore, bispecific antibodies have been used to arm in vitro polyclonal CTL populations that have been subsequently reinfused into tumor patients (Roosnek 1989, J. Exp. Med. 170, 297-302; Bolhuis 1992, Int. J. Cancer Suppl. 7, 78-81). For this “T cell arming” approach a high affinity binding to CD3 is absolutely required so that the T cells can retain the bispecific molecule on their surface until they have a chance to interact with tumor cells. However, these results can not be generalized. In different experimental settings different binding affinities might be measured.
It has to be especially emphasized that the data on T cell activation generated from TCR-peptide/MHC complexes cannot be transferred to the situation of an antibody-induced T cell activation. In the artificial situation of an antibody-induced T cell activation, the kinetics of the process may be completely different from the kinetics during the natural TCR-peptide/MHC interaction, since for example one of the major components, the peptide/MHC complex, is not present. Consequently, the kinetics are no longer determined by the TCR-peptide/MHC interaction or by the interaction of additional components like CD8 with the peptide/MHC complex.
Therefore, the available TCR-peptide/MHC T cell activation data cannot be applied to the mechanism of an antibody-induced T cell activation nor the T cell activation mechanism induced by any other biosynthetic molecule.
The CD3 complex denotes an antigen that is expressed on T-cells as part of the multimolecular T-cell receptor complex. It consists of several different chains for instance γ, δ, ε, ζ. or/and η chains. Clustering of CD3 on T cells, e.g., by immobilized anti-CD3-antibodies, leads to T cell activation similar to the engagement of the T cell receptor but independent from its clone typical specificity. Actually, most anti-CD3-antibodies recognize the CD3ε-chain.
Prior art has exemplified T cell activation events employing antibody molecules. For example, U.S. Pat. No. 4,361,544 proposes a hybrid cell line for the production of monoclonal antibody to an antigen found on normal human T cells and cutaneous T lymphoma cells and defines the antibody produced as “OKT3”. In U.S. Pat. No. 5,885,573 the murine OKT3 (described in U.S. Pat. No. 4,361,549) has been transferred into a human antibody framework in order to reduce its immunogenicity. Furthermore, U.S. Pat. No. 5,885,573 discloses specific mutations in the FcR-binding segment of OKT-3 which leads to a Glu at position 235, a Phe at position 234 or a Leu at position 234, i.e. to specific mutations in the CH2 region which are supposed to result in modified binding affinities for human FcR. In proliferation assays or in assays relating to the release of cytokines, the mutated OKT-3 antibodies disclosed in.
U.S. Pat. No. 5,885,573 appear to result in comparable cell proliferations to that observed with PBMC stimulated with the original murine OKT3 and to similar amounts of cytokines produced. Merely the mutated Glu-235 mAb induced smaller quantities of TNF-α and GM-CSF and no IFN-γ. No T cell proliferation was induced by Glu-235 mab using PBMC from three different donors at mab concentrations up to 10 μg/ml, suggesting that the alteration of the FcR binding region of this mab had impaired its mitogenic properties. T cell activation by Glu-235 mab also resulted in lower levels of expression of surface markers Leu23 and IL-2 receptor. U.S. Pat. No. 5,929,212 discloses a recombinant antibody molecule in which the binding regions have been derived from the heavy and/or light chain variable regions of a murine anti-CD3 antibody, e.g. OKT3, and have been grafted into a human framework. Similarly, U.S. Pat. No. 5,885,573 discloses the transfer of binding specificity from OKT3 into a human framework. WO 98/52975 discloses a mutated variant of the murine anti-CD3 antibody OKT3. The mutated OKT3 antibody is produced using a recombinant expression system and WO 98/52975 proposes that the mutated anti-CD3 antibody is more stable than the parental OKT3 protein during extended storage periods. U.S. Pat. No. 5,955,358 discloses a method of shuffling, at the DNA level, multiple CDR domains, either from the same or different antibodies, meaning that their order within antibody variable domains is altered to yield new combinations of binding regions.
As mentioned above, the OKT3 antibody is a mouse anti-human CD3 monoclonal antibody (mAb), derived from the murine hybridoma OKT3. It recognizes an epitope on the epsilon subunit of the human CD3 complex. OKT3 was originally described in U.S. Pat. No. 4,361,544 and U.S. Pat. No. 4,658,019; Kung 1979, Science 206, 347-349; Van Wauwe 1980, J. Immunol. 124, 2708-2713; Transy 1989, Eur. J. Immunol. 19, 947-950. Since then, OKT3 has been used as potent immunosuppressive agent in clinical transplantation to treat allograft rejection (Thistlethwaite 1984, Transplantation 38, 695-701; Woodle 1991, Transplantation 51, 1207-1212; Choi 2001, Eur. J. Immunol. 31(1), 94-106). Major draw backs of this therapy are T cell activation manifested in cytokine release due to cross-linking between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. Several publications have described alterations like humanization of OKT3 to reduce those side effects: U.S. Pat. No. 5,929,212; U.S. Pat. No. 5,885,573 and others. On the other hand, OKT3 or other anti-CD3-antibodies can be used as immunopotentiating agents to stimulate T cell activation and proliferation (U.S. Pat. No. 6,406,696 Bluestone; U.S. Pat. No. 6,143,297 Bluestone; U.S. Pat. No. 6,113,901 Bluestone; Yannelly 1990, J. Immunol. Meth. 1, 91-100). Anti-CD3-antibodies have also been described as agents used in combination with anti-CD28-antibodies to induce T cell proliferation (U.S. Pat. No. 6,352,694).
OKT3 has further been used by itself or as a component of a bispecific antibody to target cytotoxic T cells to tumor cells or virus infected cells (Nitta 1990, Lancet 335, 368-376; Sanna 1995, Bio/Technology 13, 1221-1224; WO 99/54440). Approaches up to now using antibodies as agents for recruiting T-cells have been hampered by several findings. First, natural or engineered antibodies having a high binding affinity to T-cells often do not activate the T-cells to which they are bound. Second, natural or engineered antibodies having a low binding affinity to T-cells are also often ineffective with respect to their ability to trigger T-cell mediated cell lysis.
A recently described novel approach to stimulate and/or modify T-cell response, in particular in human patients, comprise the use of single chain antibody constructs as well as bispecific molecules/bispecific antibody molecules. Such molecules and approaches are described in WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Löffler, Blood, (2000), 95, 6, 2098-2103, Brühl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56.
Antibody constructs, in particular single chain constructs and/or constructs of a bispecific formate, triggering T cell activation in the presence of a target cell are highly potent molecules in treatment of tumorous diseases, autoimmune diseases, inflammatory and infectious diseases. Such constructs are described, inter alia in WO 99/54440, wherein T-cell activation is mediated via anti-CD3 part of such antibody constructs. Bispecific single chain antibody constructs are activating T-cells only in presence of a specific target cell leading to a cytotoxic activity on the target cells. T-cell activation relates to expression of numerous genes, in particular genes encoding cytokines like IFNy, IL-2, IL-3, TGF-β, TNF-β, IL4, IL5, IL6 and GM-CSF (see Kuby, Immunology 4th edition, p. 249). It is also known that therapeutic application of antibody constructs as described in the prior art with strong T-cell activating capacity as described in the prior art also induce a strong release of cytokines.
Cancer and especially autoimmune diseases are known to be associated with release of different cytokines. Release of sIL-2R, IL-4, IL-6, IL-8, IL-10, IL-12 and TNF-alpha was shown in patients with various autoimmune disturbances such as persistent neutropenia, immune thrombocytopenia, pure red-cell aplasia, Hashimoto's thyroiditis, sicca syndrome, systemic lupus erythemathosus, systemic scleroderma (Shvidel, Hematol J. 2002, 3, 32-7). For these reasons, antibody therapy employing, inter alia, single chain constructs in patients with autoimmune diseases should not induce a further increase of the level of pro-inflammatory cytokines.
In patients with immunogenic tumors like malign melanoma or kidney cell carcinoma very few spontaneously occurring T-cells directed against tumor cells are found. The tumor specific immune response observed in patients suffering from these diseases is not sufficient to reduce or eliminate the tumor. Accordingly, it is desired to establish a therapeutic approach which leads to a reduction or elimination of the tumor without inducing severe side effects, like increased expression of soluble cytokines. Accordingly, in these patients it is desired to enhance the antigen specific immune response via activating of (an) already existing small subpopulation(s) of T-cells specific for a tumor antigen. A similar situation is observed in patients with virus infections without spontaneous recovery, for example in chronic hepatitis (like hepatitis C). Endogenous virus-specific T-cell immunity exists but is not sufficient to control the virus infection. In these cases an enhancement of this low antigen-specific T-cell response would be desirable.
Accordingly, in certain medical settings, selective and modified activation of endogenous antigen-specific T-cell population(s) is desired.
Therefore, the technical problem underlying the present invention was to provide for means and methods for pharmaceutical intervention of disorders where selective and/or modified activation of specific T-cell populations is desired and wherein the endogenous immune response of the patient to be treated has to be modified, selectively enhanced and/or “fine-tuned”. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.